Urea residue detection kit and detection method thereof
By using a specific composition of urea residue detection kit and enzyme label plate to detect urea, the absorption value of indiphenol blue products is changed at a specific wavelength, which solves the problem of low sensitivity of existing urea detection kits, and achieves high sensitivity and rapid urea residue detection, which is suitable for urea residue quality control in biopharmaceutical processes.
Patent Information
- Application Number
- CN202510542673.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-27
- Publication Date
- 2025-07-25
AI Technical Summary
The existing urea detection kit has low sensitivity, poor durability and unclear results, which is not conducive to the widespread application of urea residues in biopharmaceutical processes.
The urea residue detection kit consisting of a phosphate-EDTA buffer with a concentration of 20 to 22 mmol/L, an alkaline sodium hypochlorite solution with a concentration of 30 to 32 mmol/L, and a color-developing solution containing 0.8 to 1.0 mmol/L, was used to perform urea residue detection using an enzyme plate coupled with urease, and the urea content was measured by generating a characteristic absorption peak of the blue indiphenol blue product at 630 nm.
It realizes urea residue detection with high sensitivity, rapid detection and clear result determination, and is suitable for real-time quality control of urea residues in biopharmaceutical processes.
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Figure CN120369654A_ABST
Abstract
Description
Technical Field
[0001] This application belongs to the technical field of urea detection, and particularly relates to a urea residue detection kit and a detection method thereof. Background Art
[0002] As a representative of emerging technologies, the biopharmaceutical industry has always been the fastest-growing industry in the global pharmaceutical industry. Due to the huge development potential of biotechnology in the pharmaceutical field, genetically engineered recombinant protein drugs are widely used in various major diseases, and the market potential is huge. The Escherichia coli prokaryotic expression system is the most widely used recombinant protein expression system at present due to its clear genetic background, easy cultivation, simple operation, high expression efficiency, and low cost. Due to the too-fast expression speed of the Escherichia coli expression system and the inability of active disulfide bonds to pair correctly in time, foreign proteins often express in the form of inactive inclusion bodies in Escherichia coli. In order to obtain bioactive recombinant proteins, it is necessary to perform denaturation and renaturation operations on the recombinant proteins expressed in the form of inclusion bodies and carry out separation and purification.
[0003] Urea is a deprotonating agent that can interfere with the non-covalent interactions of proteins by forming hydrogen bonds with polar amino acid residues in proteins. This interference causes changes in the spatial structure of proteins and promotes their denaturation. In drug development and protein engineering, urea is used as a tool to study protein folding and stability.
[0004] In order to ensure protein quality, urea needs to be removed in the process. The residue of urea will not only affect the product quality but also affect the establishment of quality control methods. Therefore, it is necessary to perform quality control detection on the urea residue in the pharmaceutical process. At present, conventional urea detection kits have low sensitivity, poor durability, and unclear result display, which are not conducive to wide application. Therefore, there is an urgent need to develop a brand-new urea residue rapid detection kit with high sensitivity, good durability, and clear result display for use. Summary of the Invention
[0005] The purpose of this application is to provide a urea residue detection kit and a detection method thereof, aiming to solve the problems in the prior art that the urea detection kit has low sensitivity, poor durability, and unclear result display, which are not conducive to wide application.
[0006] To achieve the above application purpose, the technical solution adopted in this application is as follows:
[0007] In the first aspect, this application provides a urea residue detection kit, which includes the following components:
[0008] Phosphate-EDTA buffer solution with a concentration of 20 - 22 mmol / L;
[0009] An alkaline sodium hypochlorite solution with a concentration of 30 to 32 mmol / L;
[0010] A chromogenic solution containing 0.8 to 1.0 mmol / L of phenol-sodium nitroprusside;
[0011] An enzyme-labeled plate coupled with urease.
[0012] In some embodiments, the pH of the phosphate-EDTA buffer is 7.0 - 8.0.
[0013] In some embodiments, the phosphate-EDTA buffer contains disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, and EDTA.
[0014] In some embodiments, the alkaline sodium hypochlorite solution includes 0.5 mol / L sodium hydroxide and 30 mmol / L sodium hypochlorite solution.
[0015] In some embodiments, the quantitative limit of the urea residue detection kit is 2.5 - 2.7 μg / mL, and the detection sensitivity is 0.5 - 0.7 μg / mL.
[0016] In a second aspect, the present application discloses a method for detecting urea residue using a urea residue detection kit, comprising the following steps:
[0017] Provide a sample to be tested, remove proteins from the sample to be tested, and collect the supernatant of the sample to be tested;
[0018] Provide an enzyme-labeled plate coupled with urease, mix the supernatant of the sample to be tested with a phosphate-EDTA buffer for a first reaction, and then mix it with an alkaline sodium hypochlorite solution and a chromogenic solution of phenol-sodium nitroprusside for a second reaction to obtain a mixed solution;
[0019] Measure the absorbance value of the mixed solution at 630 nm, and calculate the content of urea in the sample to be tested through a standard curve.
[0020] In some embodiments, the preparation method of the enzyme-labeled plate coupled with urease includes the following steps:
[0021] Nitrate the enzyme-labeled plate to obtain a nitrated enzyme-labeled plate;
[0022] Wash the nitrated enzyme-labeled plate, perform silanization treatment, and then perform curing treatment to obtain a to-be-treated enzyme-labeled plate;
[0023] Incubate the enzyme-labeled plate with a urease solution to obtain an enzyme-labeled plate coupled with urease.
[0024] In some embodiments, the temperature of the first reaction is 36 - 37 °C, and the time is 20 - 25 minutes.
[0025] In some embodiments, the temperature of the second reaction is 36 - 37 °C and the time is 30 - 35 minutes.
[0026] In some embodiments, the standard curve is constructed using urea standard solutions with concentrations of 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml, and the obtained linear regression equation R 2 ≥ 0.99.
[0027] In a third aspect, the present application discloses a quality control method for urea residue in a biopharmaceutical process, which uses the above-mentioned urea residue detection kit or the above-mentioned method for detecting urea residue for detection to monitor the urea residue in the production process in real time.
[0028] The urea residue detection kit provided in the first aspect of the present application provides an enzyme-labeled plate conjugated with urease as a reaction bottom plate to adsorb the sample to be tested. Urea in the sample to be tested can be hydrolyzed by urease to produce ammonium ions (NH4 + ) and carbon dioxide. Under the alkaline condition provided by the phosphate-EDTA buffer solution, the chloramine generated by the oxidation of sodium hypochlorite and phenol are catalyzed by sodium nitroferricyanide to generate blue indophenol blue. The product has a characteristic absorption peak at 630 nm, and the content of urea is determined by the change in absorbance value. This detection kit has high sensitivity, fast detection speed, clear result determination, and good durability, which is conducive to wide application.
[0029] The method for detecting urea residue using the urea residue detection kit provided in the second aspect of the present application includes: providing a sample to be tested, removing proteins from the sample to be tested, and collecting the supernatant of the sample to be tested; providing an enzyme-labeled plate conjugated with urease, mixing the supernatant of the sample to be tested and the phosphate-EDTA buffer solution to carry out a first reaction, and then mixing with an alkaline sodium hypochlorite solution and a chromogenic solution of phenol-sodium nitroferricyanide to carry out a second reaction to obtain a mixed solution; measuring the absorbance value of the mixed solution at 630 nm, and calculating the content of urea in the sample to be tested through the standard curve. This method is based on the urea residue detection kit. Utilizing that urea can be hydrolyzed by urease to produce ammonium ions (NH4+) and carbon dioxide, under the alkaline condition provided by the phosphate-EDTA buffer solution, the chloramine generated by the oxidation of sodium hypochlorite and phenol are catalyzed by sodium nitroferricyanide to generate blue indophenol blue. The product has a characteristic absorption peak at 630 nm, and the content of urea can be accurately determined through the change in absorbance value; this determination method is simple and fast, which is conducive to wide application.
[0030] The quality control method for urea residue in the biopharmaceutical process provided by the third aspect of the present application uses the above urea residue detection kit or the above method for detecting urea residue for detection. Since the provided detection method is fast, has high detection sensitivity and good durability, it can monitor the urea residue in the production process in real time and can be widely used in industry. Description of the Drawings
[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments or the description of the prior art. Obviously, the following described drawings are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.
[0032] Figure 1 It is a schematic diagram of adding samples to an enzyme-linked immunosorbent assay (ELISA) plate provided by an embodiment of the present application. Detailed Embodiments
[0033] In order to make the technical problems to be solved, technical solutions and beneficial effects of the present application more clearly understood, the following further details the present application in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.
[0034] In the present application, the term "and / or" describes the association relationship of associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Wherein A and B can be singular or plural. The character " / " generally represents an "or" relationship between the associated objects before and after.
[0035] In the present application, "at least one" means one or more, and "a plurality" means two or more. "At least one (item)" or its similar expression means any combination of these items, including any combination of single item (item) or plural items (items). For example, "at least one (item) of a, b, or c", or, "at least one (item) of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.
[0036] It should be understood that in various embodiments of the present application, the magnitudes of the serial numbers of the above processes do not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.
[0037] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a" and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.
[0038] The weight of the relevant components mentioned in the specification of the embodiments of the present application not only can refer to the specific content of each component, but also can represent the proportional relationship of the weights between the components. Therefore, as long as the content of the relevant components in the specification of the embodiments of the present application is enlarged or reduced in proportion, it is within the scope disclosed in the specification of the embodiments of the present application. Specifically, the mass in the specification of the embodiments of the present application can be mass units well-known in the chemical industry such as μg, mg, g, kg, etc.
[0039] The terms "first" and "second" are only used for descriptive purposes to distinguish objects such as substances from each other, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity of the indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX can also be referred to as the second XX, and similarly, the second XX can also be referred to as the first XX. Thus, the features defined with "first" and "second" can explicitly or implicitly include one or more of such features.
[0040] The first aspect of the embodiments of the present application provides a urea residue detection kit, and the urea residue detection kit includes the following components:
[0041] A phosphate-EDTA buffer solution with a concentration of 20 - 22 mmol / L;
[0042] An alkaline sodium hypochlorite solution with a concentration of 30 - 32 mmol / L;
[0043] A chromogenic solution containing 0.8 - 1.0 mmol / L of phenol-sodium nitroprusside;
[0044] An enzyme-labeled plate conjugated with urease.
[0045] The urea residue detection kit provided by the first aspect of the embodiments of the present application, this urea residue detection kit provides an enzyme-labeled plate conjugated with urease as a reaction bottom plate to adsorb the sample to be detected. Urea in the sample to be detected can be hydrolyzed by urease to produce ammonium ions (NH4 + ) and carbon dioxide. Under the alkaline condition provided by the phosphate-EDTA buffer solution, the chloramine generated by oxidation with sodium hypochlorite and phenol are catalyzed by sodium nitroprusside to generate blue indophenol blue. The product has a characteristic absorption peak at 630 nm. By the change of the absorbance value, the content of urea is determined. This detection kit has high sensitivity, fast detection speed, clear result determination and good durability, which is conducive to wide application.
[0046] In some embodiments, the pH of the phosphate-EDTA buffer is 7.0 - 8.0. In some specific embodiments, the pH of the phosphate-EDTA buffer is selected from typical but non-limiting values such as 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, etc.
[0047] In some embodiments, the phosphate-EDTA buffer contains disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride, and EDTA.
[0048] In some embodiments, the alkaline sodium hypochlorite solution includes 0.5 mol / L sodium hydroxide and 30 mol / L sodium hypochlorite solution.
[0049] In some embodiments, the quantitative limit of the urea residue detection kit is 2.5 - 2.7 μg / mL, and the detection sensitivity is 0.5 - 0.7 μg / mL.
[0050] In some specific embodiments, the quantitative limit of the urea residue detection kit is 2.5 μg / mL, and the detection sensitivity is 0.5 μg / mL.
[0051] The second aspect of the embodiments of the present application discloses a method for detecting urea residue by a urea residue detection kit, including the following steps:
[0052] S01. Provide a sample to be tested, remove the protein from the sample to be tested, and collect the supernatant of the sample to be tested;
[0053] S02. Provide an enzyme-labeled plate conjugated with urease, mix the supernatant of the sample to be tested and the phosphate-EDTA buffer for the first reaction, and then mix with the alkaline sodium hypochlorite solution and the chromogenic solution of phenol-sodium nitroprusside for the second reaction to obtain a mixed solution;
[0054] S03. Measure the absorbance value of the mixed solution at 630 nm, and calculate the content of urea in the sample to be tested through the standard curve.
[0055] The method for detecting urea residue provided by the second aspect of the embodiments of the present application using the urea residue detection kit includes: providing a sample to be tested, removing proteins from the sample to be tested, and collecting the supernatant of the sample to be tested; providing an enzyme-labeled plate conjugated with urease, mixing the supernatant of the sample to be tested and a phosphate-EDTA buffer solution for a first reaction, and then mixing with an alkaline sodium hypochlorite solution and a chromogenic solution of phenol-sodium nitroprusside for a second reaction to obtain a mixed solution; measuring the absorbance value of the mixed solution at 630 nm, and calculating the content of urea in the sample to be tested through a standard curve. This method is based on the urea residue detection kit. Utilizing the fact that urea can be hydrolyzed by urease to produce ammonium ions (NH4+) and carbon dioxide, under the alkaline conditions provided by the phosphate-EDTA buffer solution, the chloramine generated by oxidation with sodium hypochlorite and phenol are catalyzed by sodium nitroprusside to produce blue indophenol blue. The product has a characteristic absorption peak at 630 nm. Through the change in the absorbance value, the content of urea can be accurately measured; this detection method is simple and fast, which is conducive to wide application.
[0056] In step S01, a sample to be tested is provided, proteins are removed from the sample to be tested, and the supernatant of the sample to be tested is collected.
[0057] In step S02, an enzyme-labeled plate conjugated with urease is provided, the supernatant of the sample to be tested and a phosphate-EDTA buffer solution are mixed for a first reaction, and then mixed with an alkaline sodium hypochlorite solution and a chromogenic solution of phenol-sodium nitroprusside for a second reaction to obtain a mixed solution.
[0058] In some embodiments, the preparation method of the enzyme-labeled plate conjugated with urease includes the following steps:
[0059] S021. Nitrating the enzyme-labeled plate to obtain a nitrated enzyme-labeled plate;
[0060] S022. Cleaning the nitrated enzyme-labeled plate, performing silanization treatment, and then performing curing treatment to obtain a to-be-treated enzyme-labeled plate;
[0061] S023. Incubating the enzyme-labeled plate with a urease solution to obtain an enzyme-labeled plate conjugated with urease.
[0062] In step S021, the enzyme-labeled plate is nitrated to obtain a nitrated enzyme-labeled plate. The specific steps include: treating the wells of a polyethylene ELISA plate with a newly prepared mixture of HNO3 and H2SO4 (ratio of 47:53, 250 μL / well) at room temperature for half an hour, so that NO2 groups are generated on the surface of the polystyrene well bottom plate, obtaining a nitrated enzyme-labeled plate.
[0063] In step S022, the nitrated enzyme-linked immunosorbent assay (ELISA) plate is washed, subjected to silanization treatment, and then cured to obtain the ELISA plate to be processed. The specific steps include: thoroughly rinsing the nitrated ELISA plate with deionized water to remove the excess acid in the wells. Then, the thoroughly rinsed plate is treated with a 1.5% aqueous solution of (3-aminopropyl)trimethoxysilane (pH 4.5) on a shaker at room temperature for 2 hours. Then, the plate is washed twice with deionized water and cured in a hot air oven at 60 °C for 2 hours to obtain the ELISA plate to be processed.
[0064] In step S023, the ELISA plate treated by incubation with urease solution is used to obtain the ELISA plate conjugated with urease.
[0065] The plate is thoroughly washed with methanol and dried at room temperature to remove any adhering impurities. Then, the ELISA plate to be processed is incubated with a freshly prepared 2% aqueous glutaraldehyde solution (250 μL per well) at room temperature for 2 hours. Then, it is washed with deionized water and incubated with a freshly prepared 0.1 mg / ml urease solution (250 μL per well) at 4 °C for 2 h to finally immobilize the enzyme, obtaining the ELISA plate conjugated with urease.
[0066] Furthermore, the supernatant of the sample to be measured is mixed with phosphate-EDTA buffer solution and then undergoes a first reaction.
[0067] In some embodiments, the temperature of the first reaction is 36 - 37 °C and the time is 20 - 25 minutes. In some specific embodiments, the temperature of the first reaction is 37 °C and the time is 20 minutes.
[0068] Furthermore, it is mixed with alkaline sodium hypochlorite solution and the chromogenic solution of phenol-sodium nitroprusside and then undergoes a second reaction to obtain a mixed solution.
[0069] In some embodiments, the temperature of the second reaction is 36 - 37 °C and the time is 30 - 35 minutes. In some specific embodiments, the temperature of the second reaction is 37 °C and the time is 20 minutes.
[0070] S03. Measure the absorbance value of the mixed solution at 630 nm and calculate the urea content in the sample to be measured through the standard curve.
[0071] Since urease hydrolyzes urea to produce NH4 + and CO2; under alkaline conditions, the chloramine generated by the oxidation of sodium hypochlorite and phenol are catalyzed by sodium nitroprusside to generate blue indophenol blue, and the product has a characteristic absorption peak at 630 nm. The urea content is measured through the change in absorbance value.
[0072] In some embodiments, the standard curve is constructed using urea standard solutions with concentrations of 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml, and the obtained linear regression equation R 2 ≥ 0.99. In some specific embodiments, the linear regression equation R 2 = 0.9967.
[0073] The third aspect of the embodiments of the present application discloses a quality control method for urea residue in a biopharmaceutical process, which uses the above-mentioned urea residue detection kit or the above-mentioned method for detecting urea residue for detection to monitor the residue amount of urea during the production process in real time.
[0074] The quality control method for urea residue in the biopharmaceutical process provided by the third aspect of the embodiments of the present application uses the above-mentioned urea residue detection kit or the above-mentioned method for detecting urea residue for detection. Since the above-provided detection method is fast, has high detection sensitivity, and good durability, it can monitor the residue amount of urea during the production process in real time and can be widely used in industry.
[0075] The following is illustrated with specific embodiments.
[0076] Example 1
[0077] Method for detecting residual urea using a urea residue detection kit
[0078] S01. Provide 2 ml of the original solution sample of the production process as the sample to be tested, centrifuge the sample to be tested at 12,000 revolutions per minute for 10 minutes to remove proteins, and collect the supernatant of the sample to be tested;
[0079] S02. Provide an enzyme-labeled plate conjugated with urease. The preparation method of the enzyme-labeled plate conjugated with urease includes the following steps: Treat the wells of a polyethylene ELISA plate with a newly prepared mixture of HNO3 and H2SO4 (47:53 ratio, 250 μL / well) at room temperature for half an hour to generate NO2 groups on the surface of the polystyrene well bottom plate, obtaining a nitrated polystyrene plate. Thoroughly rinse the surface of the well bottom plate with deionized water to remove the excess acid in the wells and obtain the nitrated polystyrene plate. Then, treat the thoroughly rinsed plate with a 1.5% aqueous solution of (3-aminopropyl)trimethoxysilane (pH 4.5) on an oscillator at room temperature for 2 hours, then wash it twice with deionized water, and cure it at 60 °C in a hot air furnace for 2 hours to obtain a silanized plate; then thoroughly wash the plate with methanol and dry it at room temperature to remove any adhering impurities. Then, incubate the dried silanized plate with a newly prepared 2% aqueous glutaraldehyde solution (250 μL / well) at room temperature for 2 hours. Then wash it with deionized water and incubate it with a freshly prepared 0.1 mg / ml urease solution (250 μL / well) at 4 °C for 2 h to finally immobilize the enzyme, obtaining an enzyme-labeled plate conjugated with urease;
[0080] Mix the supernatant of the sample to be tested with phosphate-EDTA buffer and incubate at 37 °C for 20 minutes, then mix with an alkaline sodium hypochlorite solution and a chromogenic solution of phenol-sodium nitroprusside and incubate at 37 °C for 20 minutes to obtain a mixture;
[0081] Among them, the preparation method of the alkaline sodium hypochlorite solution (30 mmol / L) is: Weigh 10 g of sodium hydroxide and 1.1166 / 1.861 g of sodium hypochlorite, dissolve them in water, make up the volume to 500 ml, and mix well to obtain it;
[0082] The preparation method of the phenol-sodium nitroprusside solution (0.8 mmol / L) is: Weigh 23.5 g of phenol and dissolve it in an appropriate amount of water, then weigh 0.1192 g of sodium nitroprusside and dissolve it in water, make up the volume to 500 ml, and shake well to obtain it;
[0083] The preparation method of the phosphate-EDTA buffer (20 mmol / L, pH 8.0) is to weigh 2.79 g of disodium hydrogen phosphate dodecahydrate, 0.32 g of potassium dihydrogen phosphate, 9.0 g of sodium chloride, and 1.86 g of EDTA, dissolve them in an appropriate amount of water, make up the volume to 500 ml, adjust the pH to 8.0, and shake well to obtain it;
[0084] S03. Measure the absorbance value of the mixture at 630 nm and calculate the urea content in the sample to be tested through a standard curve;
[0085] Among them, the standard curve was constructed using urea standard solutions with concentrations of 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml. Urea standard solution (1 mg / ml): Weigh accurately 10 mg of urea standard product, dissolve it with an appropriate amount of ammonia-free water, make the volume up to 10 ml, shake well to obtain it, and then dilute it to obtain standard solutions of each concentration.
[0086] During the test, take the enzyme-labeled plate with blank conjugated urease, as Figure 1 shown. A-1, A-2, and A-3 are blanks; B-1, B-2, B-3 to H-1, H-2, H-3 are standard products, and the rest are samples. Finally, place 2 quality control samples and add samples as shown in Table 1 below.
[0087] Table 1
[0088]
[0089] Example 2
[0090] Method for detecting residual urea using a urea residue detection kit
[0091] Modify "pH 8.0 of the phosphate-EDTA buffer solution" in Example 1 to "pH 7.5 of the phosphate-EDTA buffer solution", and the preparation method of the phosphate-EDTA buffer solution (20 mmol / L, pH 7.5) is as follows: Weigh 2.79 g of disodium hydrogen phosphate dodecahydrate, 0.32 g of potassium dihydrogen phosphate, 9.0 g of sodium chloride, and 1.86 g of EDTA, dissolve them with an appropriate amount of water, make the volume up to 500 ml, adjust the pH to 7.5, and shake well to obtain it.
[0092] The remaining materials and method steps are the same as those in Example 1.
[0093] Example 3
[0094] Method for detecting residual urea using a urea residue detection kit
[0095] Modify "pH 8.0 of the phosphate-EDTA buffer solution" in Example 1 to "pH 7.0 of the phosphate-EDTA buffer solution", and the preparation method of the phosphate-EDTA buffer solution (20 mmol / L, pH 7.0) is as follows: Weigh 2.79 g of disodium hydrogen phosphate dodecahydrate, 0.32 g of potassium dihydrogen phosphate, 9.0 g of sodium chloride, and 1.86 g of EDTA, dissolve them with an appropriate amount of water, make the volume up to 500 ml, adjust the pH to 7.0, and shake well to obtain it.
[0096] The remaining materials and method steps are the same as those in Example 1.
[0097] Comparative Example 1
[0098] Detection Method for Residual Urea
[0099] Provide 2 ml of the stock solution sample from the production process as the sample to be tested. Centrifuge the sample to be tested at 12,000 rpm for 10 min to remove proteins, and collect the supernatant of the sample to be tested;
[0100] Provide phosphate-EDTA buffer solution (20 mmol / L, pH 8.0). The preparation method is to weigh 2.79 g of disodium hydrogen phosphate dodecahydrate, 0.32 g of potassium dihydrogen phosphate, 9.0 g of sodium chloride, and 1.86 g of EDTA, dissolve with appropriate amount of water, make up the volume to 500 ml, adjust the pH to 8.0, and shake well to obtain;
[0101] Provide alkaline potassium iodomercurate test solution with a mass percentage concentration of 5%, potassium sodium tartrate, and ammonia-free water;
[0102] Add samples according to Table 2. After thoroughly mixing the sample and the phosphate-EDTA buffer solution, react in an incubator at 37 °C for 20 minutes, further mix with potassium sodium tartrate, alkaline potassium iodomercurate test solution, and ammonia-free water, and detect at room temperature for 15 min at 420 nm.
[0103] Table 2
[0104]
[0105]
[0106] Perform performance tests on the examples and comparative examples respectively, including comparison and analysis of linearity, detection limit, precision, and stability. The results are shown in Table 3. It can be seen that the detection limits obtained in Examples 1 to 3 are all 0.5 μg / ml. It can be seen that this method has high sensitivity and good detection effect. Furthermore, through further analysis, it can be seen that the recovery rate of Example 1 reaches 100%, the precision RSD is 0.38%, and the stability is 0.52. It can be seen that the measurement results have good stability, high reliability, and accuracy. It can be seen that the method of Example 1 is used for the detection of urea residue in biopharmaceuticals, and the results are accurate, the operation is simple, and the stability is good.
[0107] Table 3
[0108]
[0109] In summary, the urea residue detection kit provided by the embodiments of the present application uses an enzyme-labeled plate conjugated with urease as a reaction bottom plate to adsorb the sample to be tested. Urea in the sample to be tested can be hydrolyzed by urease to produce ammonium ions (NH4+) and carbon dioxide. Under the alkaline conditions provided by the phosphate-EDTA buffer solution, the chloramine generated by the oxidation of sodium hypochlorite and phenol are catalyzed by sodium nitroferricyanide to generate blue indophenol blue. The product has a characteristic absorption peak at 630 nm, and the content of urea is determined by the change in absorbance value. The detection kit has high sensitivity, fast detection speed, clear result determination, good durability, and is conducive to wide application.
[0110] The above are only the preferred embodiments of the present application and are not intended to limit the present application. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A urea residue detection kit, characterized in that, The urea residue detection kit comprises the following components: A phosphate-EDTA buffer solution with a concentration of 20 - 22 mmol / L; An alkaline sodium hypochlorite solution with a concentration of 30 - 32 mmol / L; A chromogenic solution containing 0.8 - 1.0 mmol / L of phenol-sodium nitroprusside; An enzyme-labeled plate conjugated with urease.
2. The urea residue detection kit according to claim 1, wherein The pH of the phosphate-EDTA buffer solution is 7.0 - 8.
0.
3. The urea residue detection kit according to claim 1, characterized in that, The phosphate-EDTA buffer solution contains disodium hydrogen phosphate dodecahydrate, potassium dihydrogen phosphate, sodium chloride and EDTA.
4. The urea residue detection kit according to claim 1, characterized in that In the alkaline sodium hypochlorite solution, it includes 0.5 mol / L of sodium hydroxide and 30 mmol / L of sodium hypochlorite solution.
5. The urea residue detection kit according to claim 1, characterized in that, The quantitative limit of the urea residue detection kit is 2.5 - 2.7 μg / mL, and the detection sensitivity is 0.5 - 0.7 μg / mL.
6. A method for detecting urea residue using the urea residue detection kit according to any one of claims 1 to 5, characterized in that, It includes the following steps: Provide a sample to be tested, remove proteins from the sample to be tested, and collect the supernatant of the sample to be tested; Provide an enzyme-labeled plate conjugated with urease, mix the supernatant of the sample to be tested and the phosphate-EDTA buffer solution to carry out a first reaction, and then mix with the alkaline sodium hypochlorite solution and the chromogenic solution of phenol-sodium nitroprusside to carry out a second reaction to obtain a mixed solution; Measure the absorbance value of the mixed solution at 630 nm, and calculate the content of urea in the sample to be tested through a standard curve.
7. The method according to claim 6, wherein The preparation method of the enzyme-labeled plate conjugated with urease comprises the following steps: Nitrate the enzyme-labeled plate to obtain a nitrated enzyme-labeled plate; Wash the nitrated enzyme-labeled plate, carry out silanization treatment, and then carry out curing treatment to obtain an enzyme-labeled plate to be treated; Incubate the treated enzyme-labeled plate with a urease solution to obtain an enzyme-labeled plate conjugated with urease.
8. The method according to claim 6, wherein The temperature of the first reaction is 36 - 37 °C, and the time is 20 - 25 minutes; and / or, The temperature of the second reaction is 36 - 37 °C, and the time is 30 - 35 minutes.
9. The method according to claim 6, characterized in that, The standard curve is constructed using urea standard solutions with concentrations of 0.5 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml, and the resulting linear regression equation R 2 ≥ 0.
99.
10. A quality control method for urea residue in a biopharmaceutical process, characterized in that, Use the urea residue detection kit according to any one of claims 1 - 5 or use the method for detecting urea residue according to any one of claims 6 - 9 for detection to monitor the urea residue amount in the production process in real time.